Involvement of a phosphoprotein on the zymogen granule membrane in the control of regulated exocytosis in the exocrine pancreas.

The pancreatic acinar cell is one of a number of cell types in which phosphoproteins are believed to be involved in the control of regulated exocytosis. We have examined the effects of three agents that affect secretion in the acinar cell on the phosphorylation states of proteins on the zymogen granule membrane. We show that Ca2+ and GTP gamma S, which stimulate secretion, also stimulate the phosphorylation of a protein of M(r) 45,000 (p45) on isolated zymogen granules. On the other hand, the protein kinase inhibitor genistein inhibits both secretion and phosphorylation of p45. For all three agents, p45 phosphorylation is affected over concentration ranges identical to those that affect secretion. The stimulatory effect of GTP gamma S and the inhibitory effect of genistein are also seen when the phosphorylation state of p45 on granules within permeabilized cells is examined. Ca2+, however, over the same concentration range, now causes dephosphorylation of p45. Furthermore, the time-course of this effect is similar to that of Ca(2+)-triggered secretion. Phosphorylation of p45 is exclusively on serine, with no detectable phosphorylation on either threonine or tyrosine. We propose that exocytosis in pancreatic acini is controlled at least in part through the phosphorylation/dephosphorylation of p45, with dephosphorylation acting as a trigger for exocytosis.

Effects of the immunosuppressants cyclosporin A and FK 506 on exocytosis in the rat exocrine pancreas in vitro.

1. We have examined the effects of the immunosuppressive drugs cyclosporin A (CsA) and FK 506 on exocytosis in two in vitro preparations of the exocrine pancreas-lobules and dispersed acini. 2. In lobules taken from starved rats and stimulated with the secretagogue caerulein, both CsA and FK 506, given shortly before stimulation, caused a dose-dependent inhibition of amylase secretion. In lobules from rats that had been pretreated in vivo with the protease inhibitor FOY-305 to stimulate secretion maximally, both CsA and FK 506 inhibited secretion of newly synthesized proteins, whereas only FK 506 inhibited caerulein-stimulated amylase release. 3. These different effects of the immunosuppressants on amylase release were reflected in their effects on degranulation, as revealed by electron microscopy. Control acinar cells in lobules from FOY-305-treated rats were almost completely degranulated, whereas treatment with FK 506, but not CsA, caused the accumulation of zymogen granules close to the apical plasma membrane. 4. In dispersed acini, stimulated with the cholinomimetic secretagogue bethanechol, both CsA and FK 506 reduced the secretory response, to about 45% of control; IC50 values were 50 nM and 3 nM, respectively. A similar partial inhibition of exocytosis was seen in acini permeabilized with the bacterial toxin streptolysin O and stimulated with 10 microM Ca2+. 5. These results demonstrate that the immunosuppressants cause an inhibition of exocytosis in the exocrine pancreas that is both rapid in onset and potent. The loss of the inhibitory effect of CsA on amylase release in lobules taken from FOY-305-treated rats may reveal a change in the characteristics of exocytosis as a consequence of the high level of stimulation, and also indicates that CsA and FK 506 have subtly different effects on secretion. We suggest that these drugs might be useful tools in the dissection of the molecular mechanisms of exocytosis.